The aim of experiments in this proposal is to employ a combination of genetic and molecular biological approaches for studying poliovirus replication and pathogenesis. The general approach is to use infectious cDNA clones of the poliovirus genome to isolate defined viral mutants and recombinants, and to study the replication of these viruses in cultured cells and in animals. Experiments in one section are aimed at determining the function of the 5'-upstream region of the genome, in particular a stem and loop structure that is important for RNA synthesis as well as seven upstream AUG codons whose role in viral replication are not known. Experiments are planned to explain the phenotype of a viral mutant which, in infected cells, causes a derangement of translational control such as that which might be associated with phosphorylation of eIF2 alpha. Another series of experiments employ a mouse model for poliomyelitis to identify and study viral functions essential for production of disease. Our previous studies showed that the ability of P2/Lansing to infect mice maps to the viral capsid proteins, and viral recombinants between this strain and mouse-avirulent strain will be constructed to define the precise regions of the capsid required for the mouse- adapted phenotype. The functional basis for this property will also be explored: does adaptation to mice involve selection of viral variants that recognize receptors in the mouse central nervous system? The three-dimensional structure of the P2/Lansing capsid will be determined to provide a framework for studies on the relationship between viral structure and neurovirulence. Additional information on regions of the capsid important for the mouse-adapted phenotype will come from identification of the mutations responsible for the attenuation of P2/Lansing variants resistant to neutralization with monoclonal antibodies. Finally, our studies have shown that a single base change in the 5'-up-stream region of the viral RNA, known to be partly responsible for the attenuated phenotype of the P3/Sabin vaccine, also drastically reduces P2/Lansing neurovirulence in mice. A cell line has been identified that the duplicates the effect of this mutation and will be used to elucidate the viral functions altered by the mutation. The results of these studies will contribute to our long term objective, to provide a complete description of the replication and pathogenesis of a virus that causes human disease.